We cloned the cDNA for stem cell tyrosine kinase 1 (STK-1), the human homolog of murine Flk-2/Flt-3, from a CD34+ hematopoietic stem cell-enriched library and investigated its expression in subsets of normal human bone marrow. The cDNA encodes a protein of 993 aa with 85% identity and 92% si to Flk-2/Flt-3. STK-1 is a member ofthe pe HI receptor tyrosine kinase family that includes KIT (steel factor receptor), FMS (colony-stimulating factor 1R), and platelet-derived growth factor receptor. STK-1 expression in human blood and marrow is restricted to CD34+ cells, a population greatly enriched for stem/progenitor cells. Anti-STK-1 antiserum recognizes polypeptides of 160 and 130 kDa in several STK-l-expressing cell lines and in 3T3 cells transfected with a STK-1 expression vector. Antisense oligonucleotides directed against STK-1 sequences inhibited hematopoietic colony formation, most strongly in long-term bone marrow cultures. These data suggest that STK-1 may function as a growth factor receptor on hematopoietic stem and/or progenitor cells. (17,18).Cloning of STK-1. RNA was isolated by the guanidium thiocyanate method (19). First-strand cDNA, generated from 1 &g of CD34+ total RNA with priming by random hexamers and reverse transcription by Moloney murine leukemia virus reverse transcriptase (M-MLV-RT) (GIBCO/BRL), was used as template. Degenerate oligonucleotide primers (designated HHSC-PTK1 and HHSC-PTK2), corresponding to the consensus sequences of the VIb and IX subdomains of TKs, were used for PCR amplification (11). A DNA fragment (HHSC-PTK.2A) was isolated with predicted amino acid identity of 97% when compared to the murine Flk-2/Flt-3. A CD34+ cDNA library was constructed using the Superscript plasmid system (GIBCO/BRL) and screened with this fragment. Positive clones were isolated and after sequencing (20) 5' rapid amplification of cDNA ends (21,22) was used to complete the missing 5' end of the cDNA.Reverse Transcriptase PCR (RT-PCR). Equal amounts (usually 1 ,ug) of total RNA were reverse transcribed with M-MLV-RT using random hexamers or oligo(dt)15 (Boehringer Mannheim) as primers. Aliquots of this material were used with specific primer pairs for PCR amplification. Primer pairs used for STK-1 amplification included nt 91-117 and 324-304 or 878-895 and 1557-1540. Amplification consisted of 95°C for 5 min before adding Taq polymerase (New England Biolabs), followed by cycles of 95°C for 1 min, 45°C for 1 min, and 72°C for 2 min, repeated for 35 cycles. The PCR products were electrophoresed through 1% agarose gels in TAE buffer and transferred overnight to nitrocellulose (23), processed, and probed (24).

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Genotypes and haplotypes of ADRB1, ADRB2, and ADRA2C did not significantly affect survival in metoprolol-treated or carvedilol-treated HF patients in this study. These results complement the findings of 2 similarly designed previous studies, but do not replicate an association of ADRB2 haplotypes and survival. All 3 studies differ from a survival benefit reported for bucindolol-treated homozygous ADRB1 R389 individuals. This may be attributable to a drug-specific interaction between genotype and outcome with bucindolol that does not seem to occur with metoprolol or carvedilol.